Single molecule spectroscopic techniques are becoming very sophisticated, and are now revealing information that was not possible to discern from bulk experiments. For example, polymer physics experiments with single molecules of DNA have permitted detailed study of non-equilibrium dynamics, measurement of non-linear forces of highly extended polymers, and the first direct measurement of the normal mode relaxation structure of a polymer.
Scanning probe microscopy has had a renaissance in recent years. Following the invention of the scanning optical microscope, the atomic force microscope (AFM) and near field scanning optical microscope were invented. These instruments have found many applications in biology, chemistry, physics and materials science. Recently, a far field optical interferometer was combined with an AFM in order to create a super-resolution scanning optical microscope. Contrast is generated by induced dipole-dipole interactions between the tip and sample, so that the resolution is limited only by the size of the tip. This information is encoded in the scattered electric fields and measured with an interferometer. It is an excellent method for taking extremely high resolution images of objects on a surface, but does not provide much detail for distinguishing different types of objects or chemical species. There have been some variations on the AFM, called chemical force microscopy, but these devices can only identify adhesion forces between particular ligands.
Raman spectroscopy has a long history of utility in the identification of molecular vibrational modes. In practice, Raman spectra are often used to distinguish different molecular species. However, the probability of Raman scattering is low (Raman cross sections are exceedingly small, 10.sup.-30 cm.sup.2 for CN), which limits the applicability of the technique. In 1974 it was discovered that roughened silver surfaces caused enhancements of Raman scattering by 6-7 orders of magnitude. This effect became known as surface enhanced Raman scattering (SERS) and was explained with the concept of field enhancement from classical electrodynamics. Numerous experiments and theoretical studies have been performed to confirm this picture. The key feature necessary for SERS is to have a surface roughness of characteristic size 10-100 nm, made of a material whose dielectric constant satisfies a certain resonance condition. It turns out that silver is one of the best materials for studying SERS with visible light, and many SERS studies have used silver surfaces. Both Raman and SERS Raman microprobes and microscopes have been constructed, but their resolutions are on the order of 1 mm and require a large amount of sample.
In spite of the enhancement from SERS, a single molecule will Raman scatter only a small number of photons, and photon counting detection scheme must be used. A comparable situation is found in the detection of single fluorescent dye molecules. The field of single molecule fluorescence has seen tremendous growth over the past few years, and there are now a plethora of techniques with which to detect the photons emitted from a single dye molecule. The detectors are typically avalanche photodiodes, and a wide variety of background reduction techniques have been used, including confocal optics, two photon excitation and evanescent wave excitation.